AP-4-mediated axonal transport controls endocannabinoid production in neurons

Alexandra K. Davies,Catherine G. Vasilopoulou,Marvin Ziegler,Georg H. H. Borner,Darius Ebrahimi-Fakhari,Mustafa Sahin,Fabrizio Merciai,Julian E. Alecu,Wardiya Afshar-Saber,Hellen Jumo

doi:10.1038/s41467-022-28609-w

Abstract

The adaptor protein complex AP-4 mediates anterograde axonal transport and is essential for axon health. AP-4-deficient patients suffer from a severe neurodevelopmental and neurodegenerative disorder. Here we identify DAGLB (diacylglycerol lipase-beta), a key enzyme for generation of the endocannabinoid 2-AG (2-arachidonoylglycerol), as a cargo of AP-4 vesicles. During normal development, DAGLB is targeted to the axon, where 2-AG signalling drives axonal growth. We show that DAGLB accumulates at the trans-Golgi network of AP-4-deficient cells, that axonal DAGLB levels are reduced in neurons from a patient with AP-4 deficiency, and that 2-AG levels are reduced in the brains of AP-4 knockout mice. Importantly, we demonstrate that neurite growth defects of AP-4-deficient neurons are rescued by inhibition of MGLL (monoacylglycerol lipase), the enzyme responsible for 2-AG hydrolysis. Our study supports a new model for AP-4 deficiency syndrome in which axon growth defects arise through spatial dysregulation of endocannabinoid signalling.

Highlights

The adaptor protein complex adaptor protein complex 4 (AP-4) mediates anterograde axonal transport and is essential for axon health
We and others recently discovered that adaptor protein complex 4 (AP-4) is required for the axonal delivery of vesicles containing the autophagy protein ATG9A2–5, a lipid scramblase that promotes the expansion of autophagosomal membrane[6,7]
Using very stringent data filters (false discovery rate (FDR) < 1%), we identified three proteins with a localisation shift (Supplementary Fig. 1a)—ATG9A, SERINC1 and SERINC3—which we validated as bona fide AP-4 cargoes by imaging[2]

Summary

Results and discussion

Spatial proteomics reveals DAGLB as an AP-4 cargo protein. We previously used our spatial proteomics method, Dynamic Organellar Maps, to identify cargoes of the AP-4 vesicle pathway[2]. We found that ATG9A, SERINC1 and SERINC3 were co-immunoprecipitated with the AP-4 complex[2] We reanalysed this proteomic dataset and found that DAGLB was co-enriched, along with known AP-4 vesicle proteins, while PTPN9 and LNPEP were absent (Fig. 1e). In wild-type HeLa cells DAGLB was observed in fine puncta throughout the cell, with increased density in the region of the TGN (Fig. 2a). This distribution is strikingly similar to that of ATG9A2,22. A similar increase in the amount of DAGLB at the TGN was observed in cells treated with siRNA to knock down AP-4 (Supplementary Fig. 2a, b).

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Methods

Code availability